Field of the Invention
The present invention relates to a portable automated chemical analyzing apparatus and to methods of utilizing the apparatus especially in the kinetic determination of enzyme activity and concentration of substances reactive with enzymes.
The batch analyzer of the invention provides a most convenient instrument for rapidly determining quantitative information concerning a series of chemical and especially biological samples. A series of samples of known volume can be prepared, and each analyzed in sequence and removed from the apparatus within a very short interval with ready interspacing of calibration samples.
The instrument is rapidly set up and put into operation, makes the determinations rapidly and accurately, uses a small sample size and measures true concentration. The batch analyzer of the invention does not require more than minimum dexterity to operate, is stable over extended periods and since it is fairly inexpensive and gives an individual result rapidly and conveniently, more analyses are apt to be run.
The apparatus of the invention relies on the measurement of true instantaneous rate at very early stages of the reaction before much reactant is consumed and even with gaseous reactants the reactions can be open to the atmosphere since the indicative data is collected before back diffusion of gas into the solution can influence the results.
The recorded rate sensing signal results in a sharply defined peak corresponding to apparent maximum rate which is directly proportional to initial concentration, eliminating necessity to extrapolate to true initial conditions to determine concentration.
The apparent maximum rate is obtained in a relatively short time interval of the order of 10 seconds to 10 minutes, saving analysis time, thus permitting more samples to be run in the same time interval. Direct rate sensing with the instrument of the invention is further applicable both to concentration and activity determinations and to very low levels of concentration determinations including even those previously considered trace level since they could not be effectively monitored with prior art apparatus and methods.
With reference to the enzymatic assay of glucose in blood and urine by the oxidation of glucose with glucose-oxidase enzyme, to produce hydrogen peroxide and gluconic acid, a presently available automated analyzer relies on the spectrophotometric response of the color reaction between hydrogen peroxide, peroxidase and a chromogen. The strong oxidizing agent, hydrogen peroxide can react with other reducible substances and other impurities interfere with the peroxideperoxidase system causing loss in specificity and accuracy. The assay cannot be conducted without deproteinization of blood samples or pre-purification of the urine samples and even with preliminary purification, assay of urine glucose below the trace 100 mg% level is not possible.
The batch analyzer of the invention as applied to the direct monitoring of oxygen consumed in the glucose oxidaseglucose reaction does not require preliminary purification or deproteinization of blood or urine samples, gives highly accurate results on an absolute basis and is insensitive to many impurities considered to interfere with many other analytical procedures.
The specificity and sensitivity of the analyzer of the invention in the very low ranges of glucose provides new means of identifying traumatic conditions such as diabetes at earlier stages. The ability to effectively monitor catalase activity also broadens the range of diagnostic tools available to the physician especially in recognizing the diseased states of the central nervous system.